Field of the Invention
The present invention relates generally to the fields of histology and the detection of proteins in compositions or tissues that have been preserved using an aldehyde-based cross-linking agent. Specifically, the present invention provides a method of antigen retrieval using aldehyde-scavenging agents to retrieve proteins that have been chemically modified by aldehyde fixatives contained in the compositions or tissues.
Description of the Related Art
Several fixatives are used routinely in a clinical pathology laboratory, like glutaraldehyde, formaldehyde and acetone, or other organic solvents for preservation of biological materials. The vast majority of fixation procedures, however, involve the use of aldehyde-based cross-linking agents, like formaldehyde and glutaraldehyde. The fixative solution is typically an aqueous formaldehyde solution that contains sodium phosphates, configured to provide buffering with minimal pH change following addition of a small amount of strong acid or base to pH 7.2-7.6 and an approximately isotonic solution. The fixation solution adds formaldehyde or glutaraldehyde to various groups on the proteins such as amine groups, phenol groups, thiol groups and hydroxyl groups, initially resulting in a series of reversible modifications, including imines, enamines, hydroxylmethylenes and methylene crosslinks between two amine groups. With prolonged formaldehyde fixation irreversible intermolecular and intramolecular cross-linking can occur within the protein molecules resulting in a dense network that can impair the penetration of paraffin wax or/and the access of antibody molecules. The result is that an antigen of interest may be reversibly or even irreversibly masked or an epitope may be chemically modified or destroyed by reaction with aldehyde-based fixatives.
Despite the broad use and great utility of a variety of immunohistochemical and analytical protein methods in purified proteins, protein extracts, cell or tissue sample, there is great need for further improvements. Such improvements may, for example, relate to a gentler fixation of a cell or tissue sample, to improvements in antigen retrieval or/and reproducibility of results as well as improvements to the use of antibodies to the corresponding antigen or epitope destroyed in standard procedures, like formaldehyde fixation.
Thus, there is a recognized need in the art for improved methods for unmasking proteins and retrieving antigens. Particularly, the prior art is deficient in methods and compounds that are easily formulated to reverse the aldehyde reaction with proteins in fixed tissue by reversal of imine, enamine, and methylenehydroxyl formation on amine groups in the proteins, reversal of methylene crosslinks between amino groups, and other reversible aldehyde modifications on proteins. The present invention fulfills this longstanding need and desire in the art.